Proc. Nd. Ad. Sci. USA
Vol. 72, No. 3, pp. 989-993, March 1975
Biochemical Method for Mapping Mutational Alterations in DNA with S 1
Nuclease: The Location of Deletions and Temperature-Sensitive
Mutations in Simian Virus 40*
(restriction endonucleases/single-strand cleavage)
THOMAS E. SHENK, CARL RHODES, PETER W. J. RIGBY, AND PAUL BERG
Department of Biochemistry, Stanford University School of Medicine, Stanford, California 94305
Crmlribuled by Pad Berg, Janua+y 6, 1976
ABSTRACT S1 nuclease (EC 3.1.4.X), a single-strand-
specific nuclease, can be used to accurately map the loca-
tion of mutational alterations in simian virus 40 (SUM)
DNA. Deletions of between 32 and I90 base pairs, which
are at or below the limit of detectability by conventional
electron microscopic analysis of heteroduplex DNAs, have
been located in this way. To map a deletion, a mixture of
unit length, linear DNA, prepared from the SV4Q deletion
mutant and its wild-type parent, are denatured and re-
annealed to form heteroduplexes. 91 nuclease can cut such
heteroduplexes at the nonbasepaired region to produce
fragments whose lengths correspond to the position of the
deletion. Similarly, specific fragments are produced when
S1 nuclease cleaves a heteroduplex formed from the DNAs
of SV40 temperature-sensitive mutants and either their
revertants or wild-type parents. Thus, the positions of the
nonhomolom between these DNAs can be determined.
S1 nuclease (EC 3.1.4.X) from Aspergillus oryzue degrades
single- but not double-stranded DNA (1); nevertheless, super-
helical simian virus 40 (SV 40) (2) and polyoma (3) DNAs are
converted to unit length linear molecules by this enzyme.
Presumably, this occurs because unpaired, or weakly hydro-
gen-bonded regions, susceptible to S1 nuclease, occur or can be
induced in the strained superhelical molecule. Since nicked
circular molecules appeared to be intermediates in the double-
strand cleavage, we surmised that the enzyme was capable of
cleaving the intact strand opposite or near the nick (2).
This property suggested that SI nuclease could be used to
map the location of small deletions, insertions, or, in fact, any
difference in base sequence between otherwise homologous
DNAs. For example, heteroduplex DNA molecules formed
from the complementary strands of a deletion mutant and
wild-type SV40 DNA contain a single-strand loop at a position
corresponding to the deletion (Fig. 1). If S1 nuclease can digest
this single-strand loop and subsequently cleave the intact
strand at the nick (or gap), fragments whose length cor-
responds to the position of the deletion loop should be gen-
erated. In principle, insertion mutations could also be located
this way.
Here we report that S1 nuclease can indeed be used to
locate a deletion of as few as 32 base pairs. Moreover, S1
nuclease also cleaves heteroduplex molecules formed from the
DNAs of SV40 temperature-sensitive mutants and either
their revertants or wild-type parents. If, as is likely, the base
sequence of the temperature-sensitive mutant DNA differs
Abbreviation: SV40, simian virus 40.
o Many of these experiments were reported at the Cald Spring
Harbor Symposium on Quantitative Biology, June, 1974.
from the wild-type or revertant DNA by a single base changer
this method could be useful for accurately mapping point
mutations, thereby making reliance on genetic recombination
unnecessary.
MATERIALS AND METHODS
Cells and Viruses. The origin and the procedures for
the growth of primary African green monkey kidney cells
(AGMK) and the established monkey kidney cell lines (CV-
1P and MA-134) have been described (4). All virus stocks and
virus DNA were prepared in MA-134 cells. Plaque assays were
performed on CV-1P cells. Mutants dl-808 and dl-861 are
viable deletion mutants of SV40 (5, 6). SV40 mutants ts.430
and tsD.902, as well as their wild-type parents, were obtained
from Peter Tegtmeyer (7) and Robert Martin (8), respec-
tively. Temperature-sensitive mutants were growii at 32".
Spontaneous revertants of these mutants were from plaques
that arose on cell monolayers incubated at 41".
Enzymes. SI nuclease was prepared from "Enzopharm"
powder (Enzyme Development Corp., New York) by the
procedure of Vogt (9) with minor modifications. One unit of
S1 nuclease releases 1.0 nmol of nucleotides per min at 37"
when acting on sonicated, denatured salmon sperm DNA at
pH 4.4 in the presence of 0.5 mM Zn++ and 280 mM Na+. S1
nuclease obtained from a commercial source (Miles Labora-
tories, Inc.) was also found to give good results in the types of
experiments reported here, although the "nibbling" effect (see
Results) was greater. EcoRl restriction endonuclease was pre-
pared by the procedure of Greene et al. (10). HpaI and HpaII
restriction endonucleases were prepared essentially by the
procedure of Sharp et al. (11).
DNA Substrates. SV40 DNA was extracted according to
Hirt (12) from MA-134 cells infected at a multiplicity of
<0.05 PFU per cell when >So% of the cells showed oytopathic
effect. Covalently closed viral DNA [SV40 (I)] was purified
directly from the supernatant by adding CsCl to 1.56 g/cma
and ethidium bromide to 200 pg/ml and centrifuging to
equilibrium. The band of SV40 (I) DNA was collected, and
the ethidium bromide was removed by passing the DNA
through Dowex-50 (13). Full length linear DNA or fragments
derived from it were prepared with restriction endonucleases
using published protocols (11, 14), and then the DNA was
repurified by velocity sedimentation in a neutral CsCl
gradient.
Cleavage of Hekzoduplex DNA with SI Nuclease. Hetero-
duplexes of EcoRI or HpaII endonuclease-generated linear
989
990 Biochemistry: Shed et d.
Proc. Not. Acad. Sci. USA 76 (1976)
!- !I -- i
I-
1-
/I
Single Base Change
il
Deletions or Insertions
Fro. 1.
Putative cleavages of heteroduplexes which contain
mismatched regions susceptible to S1 nuclease. A duplex DNA
with n single nick (middle) is proposed as an interrnkdiate in the
S1 nuclease cutting reaction. There is as yet no direct evidence
for such an intermediate.
SV40 DNAs were prepared by denaturing a mixture contain-
ing equal parts of two DNA species (1.5-6 pg/ml of each).in
NaOH (0.1 N). After 10 min at room temperature, the solution
was titrated to pH 7-8 with HCI, the Na+ concentration was
raised to 300 mM, and the DNA was reannealed at 68" for 3
min. The reanhealed DNA was treated with S1 nuclease (135
units/pg of DNA) in the presence of Zn++ (4.5 mM), Na+
(280 mM), and CHaCOO- (30 mM) at pH 4.4. After 30 min at
25", the reaction was terminated by raising the pH with 0.05
volume of Tris base (2 M), and by increasing the Na+ con-
centration to 500 mM. To decrease the volume and Na+
concentration of the sample prior to gel electrophoresis, the
DNA was precipitated at -20' after the addition of yeast
RNA (20 pg/ml) and 2 volumes of ethanol.
In our earlier experiments there was some cleavage by SI
nuclease of homoduplex DNA within A+T-rich regions. To
minimize the technical difficulties caused by this background,
we sought digestidn conditions that would reduce or random-
ize the location of such cleavages. Under the conditions we
adopted (4.5 mM in++, 280 mM Na+), the difference in T,
between two DNAs of substantially different base composition
is eliminated (Table 1). In 4.5 mM Zn++, Micrococcus luleus
(72% G+C) and salmon sperm {43% G+C) DNAs melt at
the same temperature (T,) and over a narrower temperature
range (AT) than was the case with our earlier conditions (0.5
mM Zn++);. With these reaction conditions inadyertent cleav-
ages are nearly random and, therefore, any fragments pro-
duced are more evenly distributed throughout the analytical
electrophoresis gels.
Gel Electrophoresti. Agarose gels (1.2%, 6 mm in diameter,
200 nim long) were prepared in Tris-borate buffer (89 mM
Tris-OH, 89 mM boric acid, 2.5 mM EDTA, pH 8.2) (10).
Samples were applied in 60 pl of Tris-borate buffer containing
sucrose (20oj, w/v). Electrophoresis was at 40 V for 17 hr.
The DNA bands were staiced with ethidium bromide and
visualized using a short wavelength ultraviolet light. The
fluorescent bands were photographed using a Vivitar orange
(02) filter and Kodak TX-135 film. The negatives were
scanned with a Joyce, Loebl and Co. microdensitometer.
RESULTS
S1 Nuclease Clews Duplex DNA at the Site of a Sillgle-
Stranded Nick. Essential to our approach of mapping deletions
is the predicted ability of S1 nuclease to digest the single-
stranded loop of a deletion heteroduplex and to cleave the
resulting nicked or gapped duplex structure at the interrup-
tion (see Fig. 1). The first expectation seemed plausible from
TABLE 1. Effect of Zn+ + on melting transitions of DNA
M. l&us Salmonsperm
DNA DNA
Buffer T,, AT. T,, AT,
S1 nuclease reaction buffer
SI nuclease reaction buffer
containing0.5mM,Zn++ 85.8 1.7 77.5 2.8
containing4.5mM Zn++ 74.5 0.8 74.2 1.6
Micrococcus lzileus and salmon sperm DNAs were heated at
approximately l"/min in the indicated buffers. Temperature was
measured with a thermocouple in a cuvette in the sample changer,
and melting transitions were monitored at 260 nm with a Gilford
model 2000 spectrophotometer. T, is the melting temperature
and AT is the breadth of the transition from I/, to '/, maximum
hyperchromicity.
the known activity of the enzyme (l), but, although earlier
experiments (2) had indicated that S1 nuclease could carry
out thesecond step, we sought more convincing evidence of
this ability. Accordingly, .SV40 DNA nicked once in either
of its two strands at the HpaII, endonuclease restriction site
(located 0.735 SV40 fractional length clockwise from the
EcoRI endonuclease-cleavage site), was converted to linear
DNA by cleavage with EcoRI endonuclease. These molecules
migrate in agarose gels as unit length linear SV40 DNA (Fig.
2A). This uniquely nicked linear DN.4 is cleaved by S1
nuclease to form two fragments, 0.26 and 0.73 SV40 fractional
length (Fjg. 2B), measured from the mobility of fragments of
known size (Fig. 2C). Because S1 nuclease appears to "nibble"
the ends of duplex DNA molecules during the course of the re-
action, the length of the fragments is slightly underestimated.
The extent of "nibbling" is difficult to determine accurately
but is about 30 base pairs or 0.006 sV40 fractional length.
Applying this correction, it is clear that the nicked DNA
molecules have been cleaved by Sl nuclease at or very near
0.735, the map coordinate of the HpaII endonuclease restric-
tion site.
S1 Nuclease Cleaves Heteroduplez DNAs at Me Sile of Dele-
lion Loops. Either naturally arising (5) or biochemically
generated (6) mutants of SV40 with deletions of the HpaII
endonuclease cleavage site are suitable model substrates for
testing the validity of the mapping procedure. DNAs from
mutants dl-808 and dZ-861, which contain deletions of about
190 and 32 base pairs, respectively (0.035 and 0.007 SV40
fractional length), as well as wild-type DNA were converted to
unit length linear DNAs by digestion with EcoRI endo-
nuclease. Electron microscopic examination of heteroduplexes
formed from d1-808 and wild-type DNA showed a barely
detectable denaturation loop 0.245 SV40 fractional length
from the nearest EcoRI endonuclease generated end (5).
Cleavage of these same heteroduplex structures with S1
nuclease produced two fragments of about 0.73 and 0.24
SV40 fractional length (15). Thus, S1 nuclease can digest the
single-stranded denaturation loop (0.035 SV40 fractional
length) and cleave the heteroduplex at that point. The dele-
tion loop in heteroduplexes formed from dl-861 and wild-type
DNA is too small to be seen in the electron microscope;
nevertheless, these molecules are readily cleaved into two
fragments whose lengths are 0.74 and 0.26 SV40 fractional
length (corrected for the ['nibbling'' effect mentioned above)
Proc. Nut. Acad. Sci. USA 78 (1976)
NO SI NUCLEASE
SI NUCLEASE-TREATED
0.53
I
IL -- c.
MARKERS
0.385
FIG. 2.
Cleavage by S1 nuclease of EcoRl endonuclease
generated linear SV40 DNA containing a singlestrand nick at
the HpaII restriction site. Relaxed circular SV40 (11) DNA, con-
taining a specific nick at the HpdI restriction site, was isolated
from a reaction in which HpaII endonuclease had converted ap
proximately 50% of the input SV40 (I) DNA to SV40 (11) and
SV40 linear (L) DNAs. The final yield of SV40 (II) DNA was
12% of the input SV40 (I) DNA. This SV40 (II) DNA (5 pg/ml),
containing a single-strand nick at the HpaII endonuclease site,
was cleaved to linear molecules with EcoRI endonuclease, and
then treated with S1 nuclease. Aliquots of the digest (0.2 pg of
DNA) were applied to gels. (A) Untreated, nicked, linear DNA.
(B) S1 nucleasetreated nicked, linear DNA. (C) Marker frag-
menta alone. These include EmRI endonuclme-generated SV40
linear DNA, fragments obtained by sequential cleavage of SV40
DNA with HpaII and EcoRI endonucleases, and fragments ob-
tained by partial cleavage of SV40 DNA with HpaI endonu-
clease. (D) Same as B with marker fryenta (0.15 pg) added.
Numbers are the length of the fragments relative to intact linear
SV40 DNA.
(Fig. 3B). Since a mixture of the corresponding homoduplexes,
formed by denaturation and renaturation of each DNA by
itself, does not yield such fragments after S1 nuclease digestion
(Fig. 3A), we may conclude that S1 nuclease can detect and
cleave a duplex DNA, preferentially, at small single-stranded
loops, and thereby permit the accurate mapping of small dele-
tions, insertions, and very likely, substitutions.
S1 Nuclease Clews Heteroduplexes Famed Between Wild-
Type, Temperature-Sensitwe, and Revertant DNAs. Since
S1 nuclease could cleave heteroduplexes at the position of
>
t
n
0
z w
2 4
I-
O
Mapping Mutations with S1 Nuclease
991
1801
MIGRATION -
FIG. 3.
Cleavage by SI nuclease of the heteroduplex prepared
from EcoRl endonuclease-generated linear DNAs of dl-861 and
its wild-type (WT) parent. The S1 nuclease reactions contained
5 pg/ml of DNA. Samples of 0.2 pg of DNA were applied to each
gel. (A) S1 nuclease-treated homoduplexes. (B) S1 nuclease-
treated heteroduplexes formed from dG861 and wild-type DNAs.
small single-stranded loops, we wished to test whether the
enzyme could cleave heteroduplex DNA at mismatches due to
single base differences. Lacking DNAs with defined single base
changes, we examined the susceptibility of heterodupleses
formed from EcoRI endonuclease-cleaved temperature-
sensitive (ts), revertant (-R) and wild-type SV40 DNAs.
Both tsAS0 and tsD202 were induced by hydroxylamine and
though the nature and number of changes in the base sequence
are not known, they are probably single base changes.
Because the DNA of tsASO and its revertant, IsASO-R, were
expected to have the lowest number of base pair differences,
we examined the S1 nuclease susceptibility of their hetero-
duplex first. As expected, the mixed homoduplexes of EcoRI
endonuclease-cut mutant and revertant DNAs did not yield
specific fragments during S1 nuclease digestion (Fig. 4A). But
the heteroduplex of the temperature-sensitive and revertant
DNA was cut to produce two easily discernible fragments of
0.68 and 0.32 SV40 fractional length (Fig. 4B). This places the
mismatch between tsAS0 and its revertant at either 0.32 or
0.68 on the SV40 map. These two alternatives could be dis-
tinguished by examining the S1 nuclease cleavage products
of the corresponding heteroduplex formed from NpaII
endonuclease-cleaved mutant and revertant DNA. In this
instance the mismatch would be expected to be in a different
location relative to the ends of the heteroduplex. Since S1
nuclease cleaved these heteroduplex DNAs, generating frag-
ments of 0.59 and 0.42 SV40 fractional length (data not
shown), the mismatch between tsASO and its revertant must
be located at 0.32 on the SV40 map.
The analysis of the S1 nucIease cleavage products of the
heteroduplex between tsASO and wild-type DNA revealed at
least one mismatched sequence (Fig. 4C). Cleavage of the
heteroduplex formed from EcoRI endonuclease-generated
linear fsAS0 and wild-type DNA with S1 nuclease yielded
Proc. Nat. Ad. Sei. USA 78 (1976)
992 Biochemistry: Shenk et d.
HOMODUPLEXE!
FIQ. 4.
Cleavage by S1 nuclease of heteroduplexes prepared
from EcoRI endonuclease-generated linear DNAs containing
putative single base mismatches. The SI nuclease reactions con-
tained 10 pg/ml of DNA. Samples of 0.7 pg of DNA were applied
to each gel. The vertical lines drawn through the DNA bands
serve to. match pairs of fragments whose sizes add up to unit
length SV40 DNA. (A) S1 nuclease-treated homoduplexes. (B)
SI nuclease-treated heteroduplexes formed from &AS0 and kASO-
R DNAs. (C) S1 nucleasetreated heteroduplexes formed from
taASO and wild-type DNAs. (D) S1 nuclease-treated hetero-
duplexes formed from kAS0-R and wild-type DNAs.
predominantly fragments of 0.62 and 0.38 SV40 fractional
length. In several earlier experiments, carried out under
different digestion conditions, S1 nuclease produced another,
apparently specific, cleavage which yielded small quantities
of fragments of 0.58 and 0.42 SV40 fractional length (15).
Only a hint of such a cleavage is seen with the present reaction
conditions (Fig. 4C). Possibly the earlier result was spurious
and there is indeed only one S1 nuclease-sensitive mismatch
in the heteroduplex of tsASO and wild-type DNA. Alterna-
tively, there is more than one mismatch in the heteroduplex of
bASO and wild-type DNA and only the site that yields the
fragments of 0.62 and 0.38 SV40 fractional length is appreci-
ably cleaved by S1 nuclease.
A decision as to whether the mismatch occurs at 0.38 or
0.62 on the SV40 map was made, as mentioned earlier, using
analogous heteroduplex molecules generated from HpaII
endonuclease-cut bAS0 and wild-type DNA; the mismatch
can be assigned to 0.38 map position (data not shown).
Heteroduplexes produced from IsASO-R and wild-type
DNAs yield two specific classes of fragments after cleavage
r 0.735
c\
i"
J 0`32 A3O-Rev
A30 Mismolch
FIQ. 5. The locations of point mutations on the SV40 chromo-
some as determined by the S1 nuclease procedure. The solid
circles on the map represent points at which Hind I1 + I11 endo-
nucleases cut, and the letters designate the fragments produced
by this cleavage (17).
with S1 nuclease (Fig. 4D). One specific cleavage generated
the same two fragments as those obtained from the hetero-
duplex prepared from bASO and wild-type DNA; the other
cleavage yielded the two fragments previously obtained with
the heteroduplex prepared from tsA3O and bASO-R DNA.
Thus, &AS0 contains at least one alteration relative to its
wild-type parent; this occurs at 0.38 on the SV40 map (Fig.
5). The alteration causing reversion of the tsAS0 phenotype
is, therefore, a second-site mutation which occurred at 0.32 on
the SV40 map. This indicates that the map coordinates 0.32 to
0.38, which is the region within which the mutant and second
site revertant differ from wild-type, lie within the A comple-
mentation group of SV40. This conclusion is reinforced by the
findings of Lai and Nathans (16), who have determined by
marker rescue experiments that the a30 mutation is located
within Hind I1 + 111 fragment H (0.375-0.43 map position)
and the L9A.28 muation (another tsA allele) occurs within
Hind I1 + I11 fragment I (0.325-0.375 map position) (see
Fig. 5). Additional experiments with other bA mutants or A
mutants resulting from deletions and insertions should better
define the physical limits of the A cistron.
The S1 nuclease mapping procedure has also been used to
locate a second SV40 cistron. Heteroduplex molecules formed
from tsD202 and a spontaneous revertant, kD802-R, were
digested with S1 nuclease as indicated above (data not shown).
A fragment 0.91 SV40 fractional length was produced by S1
nuclease cleavage of the heteroduplex formed from EcoRI
endonuclease-cut DNAs and a fragment 0.82 SV40 fractional
length from the heteroduplex of HpaII endonuclease-cut
DNAs. These results are consistent with the existence of a
nonhomology between kD.20.2 and its revertant at 0.91 on the
SV40 map.
In this instance also, the reversion does not restore the wild-
type sequence. This conclusion follows from the finding that
though S1 nuclease can cleave the heteroduplex formed from
isD.20.2-R and wild-type DNA, it fails to cleave the hetero-
duplex generated from bD.208 and wild-type DNA. Perhaps
the particular mismatch generated in the heteroduplex mode
from tsD.802 and wild-type DNA cannot be readily "recog-
nized'' or cleaved by S1 nuclease.
Here, too, the data of Lai and Nathans (16) substantiate
OUT assignment of the D cistron to a region of the physical
map containing the coordinate 0.91. They have found that
Hind I1 + I11 fragment E (map position 0.86-0.945) contains
the sequence modified by the bD.802 mutation.
Proc. Nat. Acad. Sei. USA 76 (1976)
Mapping Mutations with SI Nuclease
993
DISCUSSIONS
There Seems little doubt that S1 nuclease can be used to map
deletions and insertions (or, in fact, any change that produces
single-strand segments in heteroduplex structures) in DNA.
As with electron microscopic examination of heteroduplexes,
the method permits accurate analysis of the location and size
of these and other gross perturbations of DNA structure. Of.
particular importance, however, is the ability of the S1
nuclease-mapping procedure to detect and locate deletions and
insertions that are too small to be Seen by electron microscopy.
A most promising but still provisional conclusion is that S1
nuclease can detect and map single base changes aa well. We
are presently cautious because, though reasonable, it is still
unproven that the temperature-sensitive DNAs differ from
their revertant or wild-type DNAs by only single bases at the
sites where SI nuclease acts. Hopefully, experiments with
DNAs having known base changes at specific locations can
resolve this uncertainty.
Our limited experience with the method has already indi-
cated several shortcomings. Under the conditions used, cleav-
age of heteroduplexes with putative single base mismatches is
slower and more limited in extent than that observed with
deletion and insertion heteroduplexes. The rate-limiting step
in this cleavage is the introduction of the first nick at the site of
the mismatch, since molecules containing a single-stranded
scission are quite efficiently cleaved by the nuclease (Fig. 2).
As a consequence of the limited cleavage at the site of possible
single base mismatches, the signal (specific cutting at the mis-
match) to noise (random cutting of the heteroduplex DNA)
ratio is low. Several parameters may be tested to deal with
this problem : the conditions required to ensure perfect rean-
nealing in the formation of the heteroduplex substrates for S1
nuclease; the conditions that minimize the existence of regions
of even transient single-strandedness (e.g., reaction conditions
that eliminate or randomize transient single-strandedness due
to differences in base composition and sequence, Table 1) ; the
efficiency of S1 nuclease cleavage of mismatches involving
different base pairs occurring within different sequences
(e.g., A/G versus T/C and the influence of neighboring
stretches of high or low G+C content). A collateral problem
related to the noise or nonspecific cleavages is the possibility
that sequence heterogeneity can result during propagation of a
supposedly homogeneous DNA. With increasing experience
we should also be better able to assign error limits to the
lengths of the fragments and, therefore, to obtain more precise
assignments of the map position of mutations.
Even at its present stage of development, the SI nuclease
procedure has permitted us to map several changes in the A
and D cistrons of SV40 (Fig. 5). For example, at least one site
(0.38 on the SV40 map) distinguishes the base sequence of
tsA30 and its wild-type parent. Only one difference was
detected between the sequence in tsA3O and its revertant
tsASO-R, and that occurs at 0.32 on the map. Consistent with
this is the fact that tsASO-R differs from the wild-type at a
minimum of two map locations: 0.38 and 0.32. Thus, the re-
vertant mutation has occurred at a second site. Although we
could infer the existence of a region of nonhomology at map
position 0.91 between tsDRI2 and tsD.t?O%R and between
tsD202-R and wild-type DNAs, we have been unable to detect
a mismatch in the heteroduplex formed between tsD202 and
wild-type DNA. The reason for this is unknown and further
experiments are needed.
Some mention should be made of several ramifications of the
method. Fust, it provides an approach to genetic mapping in
systems where genetic recombination is lacking or difficult to
measure. Second, the procedure enables one to locate geno-
typic alterations that cause no detectable change in pheno-
type. Third, the ability of S1 nuclease to cleave a duplex
DNA at a mismatch may make it possible to isolate discrete
segments of a genome if that segment can be bounded by
small deletions. For example, a heteroduplex between two
different deletion mutants should yield three fragments after
S1 nuclease cleavage, one of which contains the segment
between the two mutations.
This work was supported in part by research grants from the
US. Public Health Service (GM-13235-09) and the American
Cancer Society (VC23C). T.E.S. is a Fellow of the Jane Coffin
Childs Memorial Fund for Medical Research. C.R. was supported
by funds from the CaliforniaDivision of the American Cancer Soci-
ety. P.W.J.R. is a Fellow of the Helen Hay Whitney Foundation.
Ando, T. (1966) "A nuclease specific for heat-deoatured
DNA isolated from a product of Aspergillus oryw," Bio-
dim. Biophys. Acfu 114, 158-168.
Beard, P., Morrow, J. F. & Berg, P. (1973) "Cleavage of
circular, superhelical SV40 DNA to a linear duplex by S1
nuclease," J. Virol. 12, 1303-1313.
Germond, J. E., Vogt, V. M. & Hirt, B. (1974) "Character-
ization of the singlestrand-specific nuclease S1 activity on
doublgstranded supercoiled polyoma DNA," Eur. J. BW-
chem. 43, 591-600.
Mertz, J. E. & Berg, P. (1974) "Defective SV40 genomes:
Isolation and growth of individual clones," Virology 62,
112-124.
Mertz, J. E. & Berg, P. (1974) "Viable deletion mutants of
simian virus 40: Selective isolation by means of a restriction
endonuclease from Hemophilus parainjluenzae," Proc. Nul.
Acad. Sci. USA 71,4879-4883.
Carbon, J., Shenk, T. E. & Berg, P. (1975) "A simple bio-
chemical procedure for the production of small deletions in
SV40 DNA," Proc. Nut. Ad. Sci. USA, in pres.
7. Tegtmeyer, P. (1972) "sv40 DNA synthesis: the viral
replicon," J. Virol. 10, 591-598.
8. Chou, J. Y. & Martin, R. G. (1974) "Complementation
analysis of SV40 mutants," J. Virol. 13, 1101-1109.
9. Vogt, V. M. (1973) "Purification and further properties of
singlestrand-specific nuclease from Aspergillus ory~ae,"
Greene, P. J., Betlach, M. C., Goodman, H. M. & Boyer,
H. W. (1974) "The EcoRI restriction endonuclease," in
Methods in Molecular Biology, ed. Wickner, R. B. (Marcel
Dekker, Inc., New York), pp. 87-111.
11. Sharp, P. A., Sugden, B. & Sambrook, J. (1973) "Detection
of two restriction endonuclease activities in Haemophilus
parainfluenroe using analytical agarose-ethidium bromide
electrophoresis," Biochemistry 12, 3055-3063.
12. Hirt, B. (1967) "Selective extraction of polyoma DNA from
infected mouse cell cultures," J. Mol. Biol. 26, 365-369.
13. Radloff, R., Bauer, W. & Vinograd, J. (1967) "A dye-
buoyant-density method for the detection and isolation of
closed circular duplex DNA: The closed circular DNA in
HeLa cells," Proc. Nut. Acad. Sci. USA 57, 1514-1521.
Mertz, J. E. & Davis, R. W. (1972) "Cleavage of DNA by
Rl restriction endonuclease generates cohesive ends,"
Proe. Nut. Acad. Sei. USA 69, 3370-3374.
Shenk, T. E., Rhodes, C., Rigby, P. W. J. & Berg, P. (1975)
"Mapping of mutational alterations in DNA with S1
nuclease: The location of deletions, insertions and tempera-
ture-sensitive mutations in SV40," Cold Spring Harbor
Symp. Qua&. Biol. 39, 61-67.
Lai, C. J. & Natham, D. (1974) "Mapping temperature-
sensitive mutants of SV40: Rescue of mutants by fragments
of viral DNA," Virology 60, 466-475.
Danna, K. J., Sack, G. H. & Nathans, D. (1973) "A clcavage
map of the SV40 genome," J. Mol. Biol. 78, 363-376.
1.
2.
3.
4.
5.
6.
EUT. J. Biochem. 33, 192-200.
10.
14.
15.
16.
17.